Much of what we understand about the anatomy and architecture of the immune system was revealed through exquisite experiments performed in the 1950s-1970s. These studies identified the role that anatomy played in a number of fundamental immunological phenomena including recirculation, induction of immune priming or tolerance, and the interactions of T and B cells. The recent resurgence of interest in the role of immune architecture and anatomy in basic immunological phenomena is almost entirely due to technological developments in identifying and tracking cells in vivo, not least through the ability to do this dynamically, in real time through the application of multiphoton microscopy. Here we outline the background to our own studies applying multiphoton microscopy to analysis of immune priming and tolerance, the role of adjuvants, T- and B-cell interactions, and the application of these studies in infectious and inflammatory diseases. We then describe the impact that real time in vivo imaging has had on these areas. Finally, we engage in some 'crystal ball gazing' to look at what developments in imaging are likely to occur, why they are important, and what further information these approaches may distill regarding the development of the immune response.